CN109453146B

CN109453146B - Pathogenic microorganism resisting composition and preparation method and application thereof

Info

Publication number: CN109453146B
Application number: CN201811614882.5A
Authority: CN
Inventors: 李成应; 焦伟丽; 焦晓军; 江国亮; 郭日财
Original assignee: Foshan Nanhai Eastern Along Pharmaceutical Co ltd
Current assignee: Foshan Nanhai Eastern Along Pharmaceutical Co ltd
Priority date: 2018-12-27
Filing date: 2018-12-27
Publication date: 2021-05-14
Anticipated expiration: 2038-12-27
Also published as: CN109453146A

Abstract

The invention relates to a composition for resisting pathogenic microorganisms, a preparation method and application thereof, wherein the composition for resisting pathogenic microorganisms is mainly prepared from thymol, glycerol, a solubilizer and water; the solubilizer is at least one selected from polyoxyethylene castor oil derivatives and polyglycerin fatty acid ester compositions. The thymol in the medicine is uniformly dispersed in a water-based system under the action of the solubilizer and the glycerin, the stability is good, through the synergistic interaction of the thymol and the solubilizer, pathogenic microorganisms can be effectively killed, the pathogenic microorganism infection can be prevented, inflammation caused by the pathogenic microorganism infection can be accelerated to disappear, the medicine can be used for disinfecting local tissues of animals, and preventing nipple chapping of a nipple of a milk cow, and is particularly suitable for preventing and treating mastitis of the milk cow.

Description

Pathogenic microorganism resisting composition and preparation method and application thereof

Technical Field

The invention relates to the technical field of disinfectants, in particular to a composition for resisting pathogenic microorganisms, a preparation method and application thereof.

Background

The mastitis of the dairy cattle is caused by infection of various pathogenic microorganisms, is one of the most common and serious diseases of the dairy cattle, not only affects the milk yield and causes menses loss, but also affects the quality of the milk and endangers the health of human beings. With the high-speed development of the dairy industry in China, high yield and stable yield are fundamental targets of the dairy industry, and the prevention and treatment of the mastitis of the dairy cows become important in the dairy industry.

At present, the treatment method of the cow mastitis mainly comprises an antibiotic therapy and a traditional Chinese medicine therapy. The antibiotic therapy is the direct and most effective treatment method, common medicines comprise tetracycline, streptomycin, penicillin, sulfonamides and the like, however, the long-term use of the antibiotics easily causes pathogenic bacteria to generate drug resistance, the antibiotics can remain in sick cattle bodies to pollute milk sources, and the food safety problem caused by drug residues has attracted people's extensive attention, and related regulations are distributed in many countries to limit the use of the antibiotics on livestock and poultry. The traditional Chinese medicine is from plants and animals, so that drug resistance of pathogenic bacteria is not easy to generate, the traditional Chinese medicine is metabolized in a cow body quickly and cannot remain in the cow body, and milk sources cannot be influenced.

Thymol has antibacterial, antioxidant and antiinflammatory effects, and has certain fragrance, and can be used as spice, food additive, medical medicine, oral cavity antibacterial, wound treating, anatomical specimen storing, insect repellent and antioxidant. Thymol is white crystal or colorless translucent crystal at normal temperature, and has melting point: 51.5 ℃, boiling point: 233-. At present, the medicine field is mainly used for bath foam, bactericidal disinfectant, antioxidant and the like, but the preparation and the use are generally carried out by dissolving the medicine in ethanol. Because the nipples of the dairy cows are sensitive parts of the skin, and the medicated bath solution of the nipples of the dairy cows can enter the milk and is finally eaten by people, the general medicated bath solution of the nipples of the dairy cows is required to be non-irritant and basically non-toxic.

Disclosure of Invention

Based on the composition, the composition for resisting pathogenic microorganisms is provided, and the pathogenic microorganisms can be effectively killed.

A composition for resisting pathogenic microorganism is prepared from thymol, glycerol, solubilizer and water by weight percentage; the solubilizer is selected from at least one of polyoxyethylene castor oil derivatives and polyglycerin fatty acid ester compositions.

The composition of the pathogenic microorganism has low toxicity, small irritation, no pollution to the environment and safety to people and animals, the thymol can be uniformly and stably dispersed in a composition system by the good solubilization of the specific type of solubilizer and the glycerin, and meanwhile, the specific solubilizer has a synergistic effect on the sterilization effect of the thymol, so that the composition can effectively kill the pathogenic microorganism. The thymol has the effects of bacteriostasis, antioxidation, anti-inflammation and the like, can enter a cell membrane to cause phospholipid bilayer structure disorder due to the lipophilicity of the thymol, and influence the expression of protein and a citric acid metabolic pathway, so that thalli are dead, and most pathogenic microorganisms such as escherichia coli, staphylococcus aureus, salmonella, pseudomonas fluorescens, hot necrotic hyphae and the like can be inhibited; the polyoxyethylene castor oil derivative or/and the polyglycerol fatty acid ester composition are/is used as a solubilizer, so that the dissolution of thymol can be promoted, the lipophilicity of thymol can be enhanced, and the killing effect of thymol on pathogens can be promoted; on one hand, the glycerin can be used as a solvent for dissolving thymol, and in addition, the glycerin has a good skin moistening effect and can prevent the dairy cow nipple from chapping.

In one embodiment, in the composition, the thymol content is 0.01-10%, the glycerin content is 0.1-20%, and the solubilizer content is 0.1-10% by weight percentage.

Further, the mass ratio of the glycerol to the thymol is (5-20) to 1; the mass ratio of the solubilizer to the thymol is (3-10): 1.

in one embodiment, the polyoxyethylene castor oil derivative is selected from at least one of polyoxyethylene 35 hydrogenated castor oil, polyoxyethylene 40 hydrogenated castor oil, and polyoxyethylene 60 hydrogenated castor oil; the polyglycerol fatty acid ester composition is at least one selected from polyglycerol-6 caprylate, polyglycerol-4 caprate and polyglycerol-3 cocoate.

Preferably, the solubilizer is polyoxyethylene 60 hydrogenated castor oil.

The polyoxyethylene hydrogenated 60 castor oil can enable the thymol to be well dissolved in water, so that a transparent liquid preparation is formed; but can avoid using ethanol, reduce the irritation of the composition, promote the absorption of thymol when in use, and improve the drug effect.

In one embodiment, the raw materials for preparing the composition further comprise nauclea officinalis extract, and in the composition, by weight percentage, the content of thymol is 0.5-5%, the content of the nauclea officinalis extract is 0.5-5%, the content of glycerol is 3-20%, and the content of the solubilizer is 0.1-5%.

It will be appreciated that the raw materials for preparing the composition may also include other plant extracts having anti-inflammatory effects, preferably, extracts of Nauclea officinalis in addition to extracts of Nauclea officinalis.

The nauclea officinalis extract has the effects of clearing away heat and toxic materials, diminishing inflammation, diminishing swelling and relieving pain, can promote the bactericidal action of thymol, and has the effects of preventing inflammation caused by pathogenic microorganism infection and accelerating the disappearance of inflammation caused by pathogenic microorganism infection.

In one embodiment, the mass ratio of the thymol to the nauclea officinalis extract is (1-3): 1.

In one embodiment, in the composition, the content of thymol, the content of nauclea officinalis extract, the content of glycerol and the content of solubilizer are respectively 1-5%, 0.5-1%, 5-15% and 1-5% in percentage by weight.

According to the composition for resisting pathogenic microorganisms, functional components of thymol and the nauclea officinalis extract are uniformly dispersed and dissolved in water under the solubilization of glycerin and a solubilizer to form a stable, uniform and transparent liquid preparation; the thymol and the nauclea officinalis extract are uniformly dispersed in a water-based system, so that a synergistic sterilization effect can be further achieved, the sterilization effect is improved, pathogenic microorganisms can be effectively killed, inflammation caused by pathogenic microorganism infection is prevented, and disappearance of inflammation caused by pathogenic microorganism infection is accelerated. Meanwhile, the thymol and the nauclea officinalis extract can be well dissolved in water under the action of the solubilizer, so that organic solvents such as ethanol and the like can be avoided, and the irritation of the medicine is reduced; the glycerin has good moistening effect on skin, and can prevent cracked nipple of the cow.

The invention also aims to provide a preparation method of the composition of the anti-pathogenic microorganism composition, which comprises the following steps:

according to the raw material composition of the anti-pathogenic microorganism composition, uniformly mixing the thymol, the glycerin and the solubilizer to obtain a first mixture;

adding the nauclea officinalis extract and/or the water into the first mixture, and uniformly stirring to obtain the nauclea officinalis extract.

Further, during the preparation of the first mixture, heating may be carried out at a temperature of 10 ℃ to 60 ℃ to accelerate or promote dissolution.

The preparation method is simple and safe to operate, the required equipment is simple, the obtained product is stable in quality, and industrial production is easy to realize.

It is a further object of the present invention to provide the use of a composition against pathogenic microorganisms as defined above, characterized in that it is used for killing pathogenic microorganisms in a non-therapeutic destination.

The pathogenic microorganism-resistant composition can effectively kill pathogenic microorganisms, so that the pathogenic microorganism-resistant composition can be used for preparing medicines for killing the pathogenic microorganisms and has the effect of preventing the pathogenic microorganism from infecting.

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The composition for resisting pathogenic microorganisms according to one embodiment of the invention is mainly prepared from the following raw materials in percentage by weight: 0.01 to 10 percent of thymol, 0.1 to 20 percent of glycerin, 0.1 to 10 percent of solubilizer and the balance of water; wherein the solubilizer is at least one selected from polyoxyethylene castor oil derivatives and polyglycerin fatty acid ester composition.

In one embodiment, the polyoxyethylene castor oil derivative is at least one selected from the group consisting of polyoxyethylene 35 hydrogenated castor oil, polyoxyethylene 40 hydrogenated castor oil, and polyoxyethylene 60 hydrogenated castor oil; the polyglycerin fatty acid ester composition is at least one selected from polyglycerin-6 caprylate, polyglycerin-4 caprate and polyglycerin-3 cocoate.

Preferably, the solubilizer is polyoxyethylene 60 hydrogenated castor oil. The polyoxyethylene 60 hydrogenated castor oil can make the thyme extract (i.e. thymol) be very well dissolved in water base, and the obtained mixed system has high stability; meanwhile, the polyoxyethylene hydrogenated castor oil can also improve the prevention and treatment effect of the medicaments on pathogenic microorganism infection.

In one embodiment, the mass ratio of the glycerol to the thymol is (5-20): 1, and the mass ratio of the solubilizer to the thymol is (3-10): 1.

in one embodiment, the nauclea officinalis extract is a water or ethanol extract of nauclea officinalis.

Specifically, the lignum naucleae extract is dry powder obtained by extracting leaves, branches, stems, bark and/or roots of lignum naucleae with water or ethanol solution.

Preferably, the nauclea officinalis extract is water extract dry powder of nauclea officinalis leaves.

The preparation method comprises the following steps: cutting lignum naucleae leaf, pulverizing into coarse powder, decocting in water, filtering, concentrating the filtrate, and drying under reduced pressure.

In one embodiment, the thymol is selected from at least one of thyme essential oil, oregano essential oil, and eugenol basil oil.

It will be appreciated that thymol may be added to the above drugs in purified form; alternatively, thymol-containing actives such as thyme oil or thyme extract containing thymol can be added to the above-described medicaments while ensuring that thymol is present in the medicaments of the present application at the desired effective concentration. Thyme oil or thyme extract is obtained from thyme, origanum vulgaris, and Ocimum gratissimum.

Preferably, thymol is added in the form of a thyme plant extract.

In one embodiment, the raw materials for preparing the pathogenic microorganism resisting composition further comprise a nauclea officinalis extract, and the composition is mainly prepared from the following raw materials in percentage by weight: 0.5 to 5 percent of thymol, 0.5 to 5 percent of Nauclea officinalis extract, 3 to 20 percent of glycerin, 0.1 to 5 percent of solubilizer and the balance of water.

In one embodiment, the mass ratio of thymol to the nauclea officinalis extract is (1-3): 1.

Further, the mass ratio of the thymol to the nauclea officinalis extract is (2-3): 1.

in one embodiment, the composition against pathogenic microorganisms is mainly prepared from the following raw materials in percentage by weight: 1 to 5 percent of thymol, 0.5 to 1 percent of nauclea officinalis extract, 5 to 15 percent of glycerin, 1 to 5 percent of solubilizer and the balance of water.

The composition of the pathogenic microorganism has low toxicity and small irritation, does not pollute the environment, and is safe to human and animals, wherein the thymol and the nauclea officinalis extract form a uniform mixed system under the solubilization of the solubilizer and the glycerol, and the pathogenic microorganism can be effectively killed through the synergistic action of the thymol and the nauclea officinalis extract. The thymol has the effects of bacteriostasis, antioxidation, anti-inflammation and the like, can enter a cell membrane to cause phospholipid bilayer structure disorder due to the lipophilicity of the thymol, and influence the expression of protein and a citric acid metabolic pathway, so that thalli are dead, and most pathogenic microorganisms such as escherichia coli, staphylococcus aureus, salmonella, pseudomonas fluorescens, hot necrotic hyphae and the like can be inhibited; the nauclea officinalis extract has the effects of clearing away heat and toxic materials, diminishing inflammation, diminishing swelling and relieving pain, can promote the bactericidal action of thymol, prevent inflammation caused by pathogenic microorganism infection and accelerate the disappearance of inflammation caused by pathogenic microorganism infection.

according to the raw material proportion of the anti-pathogenic microorganism composition, uniformly mixing thymol, glycerol and a solubilizer to obtain a first mixture;

adding lignum naucleae extract and/or water into the first mixture, and stirring and mixing uniformly.

The preparation method ensures that the components are fully dissolved and uniformly mixed by strictly controlling the mixing sequence of the components, and avoids the problem that certain components, particularly thymol, cannot be well dissolved in a water matrix due to improper mixing, so that the drug effect and the stability are influenced. The method is simple and safe to operate, the required equipment is simple, the obtained product is stable in quality, and industrial production is easy to realize.

Specifically, at room temperature, uniformly mixing thymol, glycerol and a solubilizer to obtain a first mixture;

adding lignum naucleae extract into the first mixture while stirring, mixing, adding water, and stirring until the lignum naucleae extract is completely dissolved.

The invention also provides an application of the pathogenic microorganism resisting composition, and the pathogenic microorganism resisting composition is used for preparing a medicament for killing pathogenic microorganisms.

The composition of pathogenic microorganisms can effectively kill pathogenic microorganisms, so that the composition can be used for preparing medicines for killing pathogenic microorganisms and has the effect of preventing pathogenic microorganism infection.

In one embodiment, the composition for resisting pathogenic microorganisms is used for preparing a medicament for killing the pathogenic microorganisms, and can be used for disinfecting local tissues of animals. Therefore, pathogenic microorganisms on the surface of local tissues of the animal can be killed, and the infection of the pathogenic microorganisms is prevented.

Specifically, the animal local tissue includes, but is not limited to, local tissues such as breast, oral cavity, vagina, uterus, ear canal, limbs, etc. It is understood that the so-called animals also include human beings, and the so-called disinfection can be disinfection of local tissues of the animals by cleaning, medicated bath, smearing, spraying and the like, so as to kill pathogenic microorganisms on the surfaces of the local tissues of the animals and prevent the pathogenic microorganisms from infecting the animals.

Preferably, the composition and the medicament for resisting the pathogenic microorganisms are particularly suitable for cleaning and disinfecting the breasts and the nipples of the dairy cows so as to prevent the occurrence of mastitis.

In one embodiment, the anti-pathogenic microorganism composition can also be used for cleaning and disinfecting pets, hatching eggs, and the like.

In one embodiment, the specific operation of disinfection may be surface cleaning, spraying, dripping, smearing, medicated bath, and the like.

The following are specific examples

Example 1

The formula of the composition for resisting pathogenic microorganisms comprises the following components: 1 wt% of thymol, 5 wt% of glycerol, 5 wt% of high-efficiency solubilizer 115C (polyoxyethylene 60 hydrogenated castor oil) and the balance of water.

(1) Accurately weighing raw material components, wherein the thyme plant extract is purchased from symris company, and the content of thymol is 99%.

(2) And uniformly mixing thymol, glycerol and the efficient solubilizer 115C according to the formula ratio at normal temperature and normal pressure to obtain a mixture.

(3) And (3) slowly adding water with the formula amount into the mixture prepared in the step (2) while stirring until the water is completely dissolved.

Example 2

The formula of the composition for resisting pathogenic microorganisms comprises the following components: 3 wt% of thymol, 10 wt% of glycerol, 1 wt% of water extract of biliary tree leaves, 3 wt% of high-efficiency solubilizer 115C (polyoxyethylene 60 hydrogenated castor oil) and the balance of water.

(1) Accurately weighing raw material components, wherein the thyme plant extract is obtained from symris corporation The phenol content is 99%, and the water extract of Nauclea officinalis is obtained from Saanergine biotechnology, Inc., and is rutin (C)₂₇H₃₀O₁₆) 3.82% in terms of equivalent) the same applies to the following examples.

(3) Adding the water extract of the nauclea officinalis in the formula amount into the mixture in the step (2).

(4) And (4) slowly adding water with the formula amount into the mixture prepared in the step (3) while stirring until the water is completely dissolved.

Example 3

The formula of the composition for resisting pathogenic microorganisms comprises the following components: 1 wt% of thymol, 0.5 wt% of water extract of biliary tree leaves, 5 wt% of glycerol, 1 wt% of high-efficiency solubilizer 115C (polyoxyethylene 60 hydrogenated castor oil) and the balance of water.

(1) Accurately weighing the raw material components.

(2) Uniformly mixing thymol, glycerol and the efficient solubilizer 115C according to the formula ratio at normal temperature and normal pressure to obtain a mixture;

(3) adding the aqueous extract of nauclea officinalis in a formula amount into the mixture in the step 2);

(4) slowly adding water with the formula amount into the mixture prepared in the step 3), and stirring while adding until the water is completely dissolved.

Example 4

Example 4 is essentially the same as example 1, except that the formulation of the drug of example 4 is: 5 wt% of thymol, 1 wt% of water extract of biliary tree leaves, 15 wt% of glycerol, 5 wt% of high-efficiency solubilizer 115C (polyoxyethylene 60 hydrogenated castor oil) and the balance of water.

Comparative example 1

Comparative example 1 is essentially the same as example 1 except that comparative example 1 replaces the high efficiency solubilizer 115C with sodium alkyl benzene sulfonate.

Comparative example 2

Comparative example 2 is substantially the same as example 1 except that the formulation of comparative example 2 is: 1 wt% thymol, 5 wt% glycerin and the balance water. During the preparation process, the obtained medicinal preparation is not completely dissolved.

Comparative example 3

Comparative example 3 is substantially the same as example 2, except that the formulation of comparative example 3 is: 3 wt% of thymol, 1 wt% of nauclea officinalis extract, 0.5 wt% of high-efficiency solubilizer 115C and the balance of water. The pharmaceutical preparation obtained was turbid.

Example 5

Example 5 is essentially the same as example 2, except that the formulation of the drug of example 5 is: 3 wt% of thymol, 10 wt% of glycerol, 1 wt% of nauclea officinalis extract, 3 wt% of polyoxyethylene 35 hydrogenated castor oil and the balance of water.

Example 6

Example 6 is essentially the same as example 2, except that the formulation of the drug of example 6 is: 3 wt% of thymol, 10 wt% of glycerol, 1 wt% of Nauclea officinalis extract, 3 wt% of polyglycerol-4-decanoate and the balance of water.

Example 7

Example 7 is essentially the same as example 2, except that the formulation of the drug of example 7 is: 0.5 wt% of thymol, 3 wt% of glycerin, 0.5 wt% of nauclea officinalis extract, 0.2 wt% of polyoxyethylene hydrogenated 60 castor oil and the balance of water.

Proof of Effect test

First, stability test

1. Test samples: the examples and comparative examples prepared compositions against pathogenic microorganisms.

2. Test protocol: the influence factor test and the accelerated test are carried out according to the annex 9001 in the pharmacopoeia of the people's republic of China (2015 edition). The specific test protocol and results are as follows:

1) influencing factors test protocol and results:

taking a test sample, respectively placing the test sample in a commercially available package under two conditions of high temperature (60 ℃) and strong light (4500 +/-500 Lx) to carry out high temperature and strong light influence factor tests, placing the test sample for 10 days, respectively carrying out thymol content detection and solution property observation on 0 day, 5 days and 10 days, and observing the stability of the test sample by taking the thymol content detection and the solution property observation as indexes, wherein the detection results are shown in the following table 1.

TABLE 1 influence factor test results

2) Accelerated test protocol and results:

the test samples were packed as commercially available and allowed to stand at 40 ℃ 2 ℃ and a relative humidity of 75% + -5% for 6 months. Samples are taken once at the end of 0 month, 1 month, 2 months, 3 months and 6 months respectively during the test period, thymol content detection and solution property observation are carried out, the stability of the test sample is inspected by taking the thymol content detection and the solution property observation as indexes, and the detection results are shown in the following table 2.

TABLE 2 accelerated test results

Second, irritation test

1. Test samples:

the pathogenic microorganism-resistant compositions of examples 1 and 2 were developed by the pharmaceutical company, Australian Dragon, Oriental, of south China sea, Foshan; 0.9% sodium chloride injection, specification: 500mL, lot A10082202, produced by Guizhou Tiandi pharmaceutical Co., Ltd.

2. Test animals:

new Zealand rabbits, 32, normal grade, weighing about 2kg, 24 females 8 males, were provided by the Guangdong provincial medical laboratory animal center. After the animals are bought, the animals are bred for 3 days in an environment-adaptive way, natural illumination is adopted, complete rabbit feed is fed during the test period, drinking water is sufficient and unrestricted, the room temperature is controlled to be 26 ℃, and the relative humidity is 40% -70%.

3. Design of experiments

1) Skin irritation test

The 16 new zealand rabbits were randomly divided into a complete skin group (group i) and a damaged skin group (group ii), 4 animals per group, each half male and female, and half of the animals were randomly selected for the animal tests of examples 1 and 2. In the first group of experiments, 24h before the start of the test, hairless areas with the area of 3cm multiplied by 3cm are shaved at the symmetrical parts on the two sides of the animal spine by using an electric shaver, and after the skin is depilated, whether the haired areas are injured due to depilation is checked, and the skin injury is not subjected to the irritation test of intact skin. The group II damaged skin is produced by cutting the dehaired and sterilized skin into epidermis layer of skin with 1cm interval in the shape of "#" with disposable sterilized No. 7 needle, and the damaged degree of the skin on the left and right sides is basically the same with the degree of blood leakage. The left side is the area for example 1, 0.5m L is applied directly to the dehaired skin on the left side, then covered with two layers of gauze (2.5cm x 2.5cm) and a layer of cellophane or similar, secured with a non-irritating adhesive tape and bandage, and the right side is the saline control area. The application time is at least 4h, after the application is finished, the tested object is removed and the administration position is cleaned by warm water or non-irritant solvent. For 3d, erythema and edema, whether pigmentation, bleeding spots, rough skin or thin skin, and the time of occurrence and time of regression were observed and recorded 1h after each removal of the drug and before reapplication, and scored with reference to the skin irritation response scoring criteria of table 3 below, using the same right-side self-contrast. After the last coating, the condition of erythema, edema and the like at the coating part is visually observed and recorded 30-60 min, 24h, 48h and 72h after the medicine is removed. For the multiple-dose skin irritation test, the mean values of each group were calculated for each observation time point, and then the mean values of each animal per day for the observation period were calculated, and the irritation intensity was evaluated according to table 4 below.

TABLE 3 skin irritation response Scoring criteria

TABLE 4 evaluation criteria for skin irritation intensity

Score value	Evaluation of
		0～0.49	Has no irritation
0.5～2.99	Mild irritation
		3.0～5.99	Moderate irritation
6.0～8.0	Strong irritation

2) Vaginal mucosa irritation test

12 female New Zealand rabbits were divided into 1 group infected with virus, 2 groups infected with virus and a control group, each group consisting of 4 rabbits. Groups 1 and 2 were vaginally perfused with 2mL of the anti-pathogenic microorganism composition of example 1 and 2, respectively, and control animals were treated with saline. 24h after infection, animals are euthanized by air embolism method, the whole vagina is taken out after laparotomy, the vagina is longitudinally split, and whether congestion, edema and other manifestations exist or not is visually observed. Fixing vagina with congestion and edema in 10% formalin solution for over 24 hr, selecting tissues at two ends and 3 central parts of vagina, making into slices, HE staining, and performing histopathological examination. Scoring is performed according to the mucosal irritation response scoring standard of the following table 5, the mucosal irritation response scores of each animal at each observation time are added to obtain a total score, and the total of the scores of one group is divided by the number of the animals to obtain a final score. The stimulation intensity was graded according to the mucosal stimulation intensity grading standard of table 6 below to judge the degree of stimulation.

TABLE 5 vaginal mucosal irritation response Scoring criteria

TABLE 6 evaluation criteria for vaginal mucosal irritation intensity

Mean value of	Evaluation of
		0～0.4	Has no irritation
0.5～1.5	Mild irritation
		1.51～2.5	Moderate irritation
＞2.5	Severe irritation

4. Test results

1) Results of skin irritation test New Zealand rabbits were observed daily on the skin administration site during the 3d administration period and within 3d after the end of administration. The results showed that no erythema and edema formation was observed in the skin (left side) of the administration site of example 1 and example 2 and the normal saline control site (right side) of the intact skin group and the damaged skin group, and the score was 0 according to the prescribed score standard; abnormal reactions such as pigmentation, drug-induced bleeding and rough skin were not observed. The activity, ingestion, drinking, defecation, urination, weight and appearance of the tested animals are not abnormal. According to the evaluation standards of skin reaction scores and reaction intensity of related skin irritation tests in China, example 1 has no irritation reaction to both intact skin and damaged skin of New Zealand rabbits, and the specific results are shown in the following table 7.

Table 7 example 2 evaluation of skin irritation in new zealand rabbits

2) And vagina mucosa irritation test results during the period of medication, no abnormality is found in the general conditions of the New Zealand rabbits of the toxicity-infected group 1, the toxicity-infected group 2 and the control group, and no obvious congestion, red swelling and abnormal secretion outflow are found in the vagina opening. The taken vaginal tissues are observed by naked eyes, each group does not have congestion, edema and bleeding points of the vaginal mucosa, the stimulation of the vaginal mucosa is averagely divided into 0, the stimulation intensity of the anti-pathogenic microorganism compositions of the examples 1 and 2 to the disposable vaginal mucosa of the New Zealand rabbits is nonirritant according to the national relevant evaluation standards of the stimulation response score and the response intensity of the vaginal mucosa, and the specific results are shown in the following table 8. No abnormalities were detected by histopathology of vaginal mucosa.

TABLE 8 EXAMPLE 1 intensity of the macroscopic irritation response to vaginal mucosa of New Zealand rabbits

The results show that the anti-pathogenic microorganism compositions of examples 1 and 2 have no irritation to skin and vaginal mucosa, and suggest that the application safety of the invention is good, the clinical use is safe, the invention meets the administration requirement of breast injection, and the invention is worthy of further popularization and application in clinic.

Third, clinical curative effect test

1. Test samples: the pathogenic microorganism-resistant compositions of examples 1 to 7 and comparative examples 1 to 3.

2. Test animals:

the total 120 clinical mastitis natural morbidity cases of a large dairy cow breeding plant in Hebei are selected. Obvious body temperature and general symptoms and poor appetite; redness, swelling, and hard mass in the mammary area; palpation has a hot, painful response; the milk has flocculus, clot or is water sample; SCC (somatic cell) more than 20 ten thousand/mL in the milk mixture, and the CMT (California mastitis detection method) tests strong positive; reduced milk production, and other clinical mastitis symptoms.

3. Test method

1) The administration method comprises the following steps: dividing the milk into 12 groups, taking 1-10 groups as test groups, cleaning the breast area after milking the affected cattle in the morning and evening, carrying out medicated bath disinfection on the nipples by using the test drug of embodiment 1, infusing 100mL of corresponding test drug embodiment into each affected breast area of the test drug group every 12h, and continuously administering for 7 days; 11 groups are prevention control groups, milk areas are cleaned after milk expressing of sick cows in the morning and evening, the nipple medicated bath disinfection is carried out by using the test drug of the embodiment 1, 100mL of the corresponding test drug of the embodiment 1 is filled into each sick milk area every 12h in the test drug group, the drug is continuously applied for 7 days, and the nipple medicated bath disinfection is not carried out by using the embodiment 1 after 7 days; 12 groups were control groups and no drug group was administered.

2) And evaluation index of therapeutic effect

A. And (3) curing: the general symptoms disappear, and the appetite returns to normal; the symptoms of red, swollen, hot and painful affected breast areas are completely disappeared, the appearance of the breast milk is recovered to be normal, and the lactation yield is obviously increased; SCC in milk is less than 20 ten thousand/mL; pathogenic bacteria were not detected in the milk samples in the milk area.

B. The method has the following advantages: the general symptoms basically disappear, and the appetite returns to normal; the symptoms of red, swollen, hot and painful affected mammary area are obviously improved or relieved; the appearance of the milk is basically normal, and the milk yield is recovered to a certain degree; SCC is reduced to some extent in milk; the pathogenic bacteria in the milk sample in the milk area are less than 1000 cfu/mL.

C. And (4) invalidation: the general symptoms do not disappear or even worsen; the symptoms of the affected breast area are not obviously improved or even aggravated; the appearance of milk is more abnormal, the milk yield is not recovered and even the lactation is stopped; or repeating the treatment within two weeks after stopping the medicine; there was essentially no change in SCC in milk; the number of pathogenic bacteria in milk samples in the breast area is more than 1000 cfu/mL.

3) Monitoring of recurrence rate

And (4) monitoring the clinical symptoms and milk-like pathogenic bacteria in the breast area within 1 week after the medicine is stopped for the cured dairy cow.

4. And (3) test results:

1) and the result of evaluation of therapeutic efficacy

2) Results of monitoring recurrence rate

Observation is carried out within 1 week after the medicine is stopped, 9 cured cows in 2 groups (example 2) and 4 groups (example 4) of test groups have no relapse, the other 1 cured cows have relapses, the cows after the cure in examples 1 and 3 have 2 relapses, the cows after the cure in examples 5 and 6 have 3 relapses, and the cows after the cure in example 7 have 4 relapses; the cows cured in the comparative examples 1 and 2 both relapse, the cure rate is 0, and only 1 cow cured in the comparative example 3 has no relapse. In group 11 (prevention control group), the medication of the example 1 is not continuously used for carrying out the teat medicated bath disinfection within one week after the medication is stopped, and the recurrence rate of the cured cows is 5; in the 12 th group (blank control group), the pathogenic microorganism composition is used for treating within 1 week after the test is stopped, and the clinical symptoms of mastitis are obviously improved after the pathogenic microorganism composition of the embodiment 1 is used for 1 week, and the effective rate reaches more than 80%.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A composition for resisting pathogenic microorganism is characterized by being prepared from thymol, glycerol, solubilizer, Nauclea officinalis extract and water;

in the composition, according to weight percentage, the content of thymol is 3% -5%, the content of the nauclea officinalis extract is 0.5% -5%, the content of glycerin is 10% -15%, the content of the solubilizer is 3% -5%, and the balance is water;

The solubilizer is selected from at least one of polyoxyethylene castor oil derivatives and polyglycerin fatty acid ester compositions;

the polyoxyethylene castor oil derivative is at least one selected from polyoxyethylene 35 hydrogenated castor oil, polyoxyethylene 40 hydrogenated castor oil and polyoxyethylene 60 hydrogenated castor oil; the polyglycerol fatty acid ester composition is at least one selected from polyglycerol-6 caprylate, polyglycerol-4 caprate and polyglycerol-3 cocoate.

2. The pathogenic microorganism-resistant composition according to claim 1, wherein the thymol is selected from at least one of thyme essential oil, oregano essential oil, and clove basil oil.

3. A composition according to claim 1, characterised in that the solubilising agent is polyoxyethylene 60 hydrogenated castor oil.

4. The pathogenic microorganism-resistant composition according to claim 1, wherein the mass ratio of thymol to the nauclea officinalis extract is (1-3): 1.

5. The pathogenic microorganism-resistant composition according to claim 1, wherein the mass ratio of thymol to the nauclea officinalis extract is (2-3): 1.

6. The composition of claim 1, where the composition includes, by weight, 3% of thymol, 1% of nauclea officinalis extract, 10% of glycerin, 3% of solubilizer, and the balance water.

7. A method of preparing a composition of an anti-pathogenic microorganism as claimed in any one of claims 1 to 6, characterised by the steps of:

8. Use of a composition according to any one of claims 1 to 6 for combating pathogenic microorganisms, in the manufacture of a medicament for killing pathogenic microorganisms.